March 2007
Volume 48, Issue 3
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Visual Neuroscience  |   March 2007
α2 Adrenergic Receptor–Mediated Modulation of Cytosolic Ca++ Signals at the Inner Plexiform Layer of the Rat Retina
Author Affiliations
  • Cun-Jian Dong
    From the Department of Biological Sciences, Allergan Pharmaceuticals, Inc., Irvine, California.
  • Yuanxing Guo
    From the Department of Biological Sciences, Allergan Pharmaceuticals, Inc., Irvine, California.
  • Larry Wheeler
    From the Department of Biological Sciences, Allergan Pharmaceuticals, Inc., Irvine, California.
  • William A. Hare
    From the Department of Biological Sciences, Allergan Pharmaceuticals, Inc., Irvine, California.
Investigative Ophthalmology & Visual Science March 2007, Vol.48, 1410-1415. doi:10.1167/iovs.06-0890
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      Cun-Jian Dong, Yuanxing Guo, Larry Wheeler, William A. Hare; α2 Adrenergic Receptor–Mediated Modulation of Cytosolic Ca++ Signals at the Inner Plexiform Layer of the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(3):1410-1415. doi: 10.1167/iovs.06-0890.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Compelling evidence suggests that α2 agonists, such as brimonidine, protect retinal ganglion cells (RGCs) from injury in a wide range of animal models. However, the mechanism of action for this protection and the physiological role of the α2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the α2 adrenergic system in the modulation of cytosolic Ca2+ signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place.

methods. Functional Ca2+ imaging at the inner plexiform layer (IPL) and outer plexiform layer (OPL) of living rat retinal slices was conducted with a high-speed confocal system. The relative changes of cytosolic free Ca2+ were monitored with the fluorescent Ca2+ dye fluo-4. The Ca2+ signal was elicited by membrane depolarization produced by a high K+ (40 mM) Ringer solution that was delivered rapidly and briefly to the test regions of the retinal slice by a custom-made multichannel local perfusion system.

results. A brief application (8 seconds) of high K+ Ringer elicited a robust cytosolic Ca2+ increase at the IPL and OPL. In both cases, this Ca2+ signal was eliminated by nimodipine, a selective L-type voltage-gated Ca2+-channel blocker, or when the extracellular Ca2+ in the Ringer was replaced with equal molar EGTA. At IPL, the Ca2+ signal was also suppressed in a dose-dependent manner by brimonidine and other α2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic α2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the α2 receptor agonists had no effect on the high K+ Ringer-elicited cytosolic Ca2+ signal at OPL. Blocking the N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K+–elicited Ca2+ signal by approximately 20% at IPL. D-AP5 had no effect on the Ca2+ signal at OPL.

conclusions. These findings provide the first direct evidence of α2 receptor–mediated modulation of L-type Ca2+ channel activity in the CNS (the retina is part of the CNS). This α2 modulation appears to occur at the IPL but not at the OPL of the retina. These findings suggest that a physiological function of the retinal α2 system is the regulation of synaptic transmission at IPL and that brimonidine and other α2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca2+ either in RGCs, in their presynaptic cells, or in both.

Retinal ganglion cells (RGCs) are the output neurons that carry visual information to higher visual centers of the brain. They are particularly vulnerable to injury in certain eye diseases such as glaucoma. 1 RGCs receive inputs from bipolar cells and amacrine cells and perhaps even glial cells at the inner plexiform layer (IPL), a synaptic region composed primarily of the neural processes of bipolar, amacrine, and ganglion cells. The other synaptic region of the retina, the outer plexiform layer (OPL), is the site of synaptic communication between photoreceptors and second-order neurons—the bipolar cells and horizontal cells. 2  
Membrane depolarization–elicited increases in the cytosolic free Ca2+ in neurons are, in most instances, essential for neurotransmitter release from presynaptic terminals. However, compelling evidence indicates that under disease conditions, an abnormal elevation of intracellular free Ca2+ can lead to neuronal cell injury and contribute to the pathophysiology of acute and chronic neurologic disorders. 3 4 5  
The α2 adrenergic receptors are G-protein–coupled receptors (GPCRs). 6 It has been shown in a number of previous studies that α2 agonists are neuroprotective in the retina. For example, brimonidine (UK14304), a classic α2 agonist, protects RGCs in animal models of glaucoma, 7 acute retinal ischemia, 8 9 10 11 and optic nerve injury. 12 This neuroprotective action of brimonidine appears to be retinal in origin and is unrelated to its IOP (intraocular pressure)-lowering effect. Retinal α2 receptors have been demonstrated in a number of mammalian species, including the rat and human. 13 14 15 However, the mechanism that underlies neuroprotection of RGCs by α2 agonists in these animal models is not well understood, primarily because the physiological function of the retinal α2 adrenergic system is largely unknown. 
In this study, we conducted functional Ca2+ imaging at the IPL and OPL of living rat retinal slices with the use of a high-speed confocal imaging system. A major goal of this work was to explore the role of the α2 adrenergic system in modulating intracellular Ca2+ signaling at retinal synaptic layers, particularly at the IPL, where communication between RGCs and their presynaptic cells takes place. 
Methods
Preparation of Living Retinal Slices
The present study was conducted in accordance with guidelines outlined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by an institutional animal care and use committee. Rat retinal slices were prepared using procedures similar to those described previously. 16 Briefly, Brown Norway rats were deeply anesthetized using intramuscular injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). After enucleation of both eyes, animals were humanely killed immediately with intracardial injection of (120 mg/kg; Eutha-6, Western Medical Supply, Arcadia, CA). Retinas were carefully isolated, and a small piece of retina (approximately 2 × 6 mm) was placed vitreal side down on a piece of black filter paper (catalog no. habp04700; Millipore, Bedford, MA). The retina and filter paper were then radially sliced at approximately 250-μm intervals. Retinal slices were carefully transferred to recording chambers and were securely positioned, after a 90° turn, by placing the filter paper to which the retinal slices were attached on two tracks of vacuum grease so that all retinal layers, including OPL and IPL, could be viewed and imaged with a fixed-stage microscope (BX50WI; Olympus America Inc., Melville, NY). 
Solutions and Test Agents
The dye-loading medium was prepared using Hanks balanced salt solution without sodium bicarbonate but with 20 mM HEPES and 2 μM fluo-4 AM (Molecular Probes, Eugene, OR). Final pH was adjusted to 7.4, with 1 M NaOH. 
The bathing medium (rat Ringer) contained 120 mM NaCl, 3 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 0.5 mM KH2PO4, 10 mM glucose, and 26 mM NaHCO3. The Ringer was bubbled continuously with 95% O2 and 5% CO2. The high K+ Ringer was prepared by replacing 37 mM NaCl in the normal rat Ringer with equal molar KCl, yielding a total K+ concentration of 40 mM. The 0 Ca2+ Ringer was prepared by replacing CaCl2 with equal molar EGTA. 
The following were purchased for the study: brimonidine (UK 14,304), yohimbine, rauwolscine, and nimodipine (Sigma, St. Louis, MO); medetomidine (Tocris, Ellisville, MO), and atipamezole (Antisedan; Novartis Animal Health, Basel, Switzerland). 
Loading Membrane Permeable Fluorescent Ca2+ Dye and Confocal Ca2+ Imaging
After retinal slices were mounted in the perfusion chambers, the chambers were filled with dye-loading medium and placed on a shaker (model 1304; Laboratory-line Instruments, Melrose Park, IL) for 50 minutes at room temperature. One of the chambers containing dye-loaded retinal slices was mounted on the microscope stage. Slices were continuously perfused with normal rat Ringer through multichannel bath and local perfusion systems. Flow rates were 3.5 and 0.3 mL/min for the bath and local perfusion systems, respectively. Imaging experiments were conducted at room temperature (approximately 20°C). 
Cytosolic Ca2+ signals were elicited by a brief (8-second) local perfusion of the high K+ Ringer that perfused the entire retinal slice being imaged. The light path shutter, high K+ perfusion, and Ca2+ imaging were precisely controlled (P-Clamp 8 software; Molecular Devices Corp., Sunnyvale, CA). Ca2+ imaging was conducted with a spinning disc confocal system (Nipkow; Solamere Technology Group, Salt Lake City, UT) equipped with a high-sensitivity, high-speed intensified charge-coupled device (CCD) camera (XR/Mega 10; Stanford Photonics Inc., Palo Alto, CA). Retinal Ca2+ images were acquired at 2 to 4 frame/s through a 60× long-working distance water immersion objective (LUMPlan FI 60×/0.90 W; Olympus America Inc, Melville, NY). 
Data Analysis
Imaging data were first measured and analyzed with QED software (MediaCybernetics, Silver Spring, MD). The measured data were then further analyzed and plotted with Origin (OriginLab, Northampton, MA) and Microsoft Excel. Statistical data were expressed as mean ± SD. 
Results
High K+ Elicited Intracellular Ca2+ Signal at IPL
In order to explore the role of the α2 adrenergic system in modulation of intracellular Ca2+ signaling at IPL, we have developed a method for functional Ca2+ imaging in living mammalian retinal slice, an ex vivo preparation that has many properties resembling those seen in vivo. In the acutely prepared rat retinal slice (Fig. 1A) , all major retinal layers, including IPL and OPL, are clearly visible and accessible for functional imaging. The yellow rectangle encloses the area in which intracellular Ca2+ signals were measured. 
We used local perfusion of a high K+ (40 mM) Ringer solution to produce membrane depolarization of IPL cell processes. Figure 1Bshows that an 8-second application of high K+ Ringer elicited a robust intracellular Ca2+ signal at IPL (red trace). This signal was completely abolished during perfusion with 0 Ca2+ Ringer (green trace). The Ca2+ signal was fully recovered after re-perfusion with normal Ca2+ solution (blue trace). This high K+-elicited Ca2+ signal was also virtually abolished after application of normal Ringer containing 10 μM nimodipine, a selective L-type Ca2+ channel blocker (Fig. 1C) . Thus, these results indicate that the high K+-elicited elevation of cytosolic free Ca2+ was produced mainly by Ca2+ influx through L-type, voltage-gated Ca2+ channels on the plasma membrane. 
α2 Receptor-Mediated Suppression of High K+-Elicited Ca2+ Signal at IPL
Next, we tested whether brimonidine could modulate this depolarization-induced intracellular Ca2+ increase. After a high K+-elicited Ca2+ response was recorded under control conditions (Fig. 2A , red trace), the slices were superfused with normal Ringer solution containing 600 nM brimonidine for 3 minutes before the high K+ solution (containing the same concentration of brimonidine as the bath solution) was applied. Brimonidine treatment abolished the high K+-elicited Ca2+ signal (Fig. 2A , green trace). The brimonidine effect was reversible. Full recovery of the high K+-elicited Ca2+ signal was usually observed after 3- to 5-minute washing with the brimonidine-free Ringers (Fig. 2A , blue trace). Brimonidine pretreatment did not seem to have a significant effect on the basal Ca2+ signal. Figure 2Bshows the dose-response relationship for brimonidine block of the high K+-elicited Ca2+ signal at IPL. The suppressive effect of brimonidine could be completely blocked by coapplication of either yohimbine or rauwolscine, two classic α2 adrenergic receptor antagonists (Figs. 2C 2D)
To further characterize the retinal α2 receptors, we also tested another widely used α2 receptor agonist, medetomidine. Figure 3Ashows that medetomidine suppressed the high K+-elicited Ca2+ signal at IPL, an effect that could also be completely blocked by yohimbine or atipamezole (a more potent and selective α2 antagonist than either yohimbine or rauwolscine). Figure 3Bshows the dose-response relationship of the medetomidine effect. Although medetomidine appeared to be more potent than brimonidine (lower IC50 than that of brimonidine), the maximal block of IPL Ca2+ signal was only approximately 75%, a result consistent with medetomidine acting as a partial agonist. Brimonidine is able to block 100% of the IPL Ca2+ signal and appears to act as a full agonist at the α2 receptor in this case. 
Lack of Suppressive Effect of Brimonidine on High K+-Elicited Ca2+ Signal at OPL
In addition to IPL, photoreceptors communicate with bipolar cells and horizontal cells at another synaptic layer in the retina, the outer plexiform layer (OPL). Therefore, we were interested in determining whether a similar α2 modulation of depolarization-induced Ca2+ signals operates at OPL as well. Application of high K+ Ringer solution elicited a robust Ca2+ signal at OPL (Fig. 4A)and IPL (Fig. 4B)in the same retinal slice. As seen for IPL, the Ca2+ signal at OPL could also be eliminated by 0 Ca2+ Ringer and nimodipine (Fig. 4C) , indicating that it is also produced predominantly by Ca2+ influx through the L-type, voltage-gated Ca2+ channels. However, the application of brimonidine had no effect on the high K+-elicited Ca2+ signal at OPL. In the same slice, a typical brimonidine-induced suppression of the Ca2+ signal was observed at IPL (Fig. 4C , third pair from left). These results demonstrate that brimonidine suppression of the depolarization-induced Ca2+ signal is region specific: it potently suppresses the Ca2+ signal at IPL but not at OPL. 
High K+ depolarization–induced Ca2+ influx through L- and other types of Ca2+ channels may trigger glutamate release from photoreceptor and bipolar cell synaptic terminals. Glutamate may activate NMDA receptors, which have a high Ca2+ permeability and are expressed predominantly, if not exclusively, on the dendrites or processes of third-order retinal neurons, particularly ganglion cells. If the high K+-elicited Ca2+ signal is mediated mainly by NMDA receptors (secondary Ca2+ signal) and if the α2 agonists also modulate NMDA receptor activity, this could explain the observed region-specific α2 modulation of high K+-elicited Ca2+ signal. We used a selective NMDA receptor antagonist, D-AP5, to test this possibility. At a concentration of 50 μM, D-AP5 completely blocked NMDA-induced whole-cell currents in retinal neurons (data not shown) and did have a small effect on the high K+-elicited Ca2+ signal at IPL but not at OPL. It reduced the signal by 17% ± 6% (Fig. 4C , right pair). Thus, suppression of the high K+-elicited Ca2+ signal at IPL by α2 agonists appeared to have been caused mainly by the modulation of L-type Ca2+ channels. 
Discussion
Our results have provided the first direct evidence in the retina for α2 receptor–mediated modulation of cytosolic free Ca2+ signal induced by membrane depolarization. To our knowledge, this is also the first evidence of α2 modulation of L-type Ca2+ channels in the CNS. We have demonstrated that this α2 modulation occurs only at the IPL, not at the OPL. Because the IPL consists primarily of neural processes of RGCs and their presynaptic cells, this unique region-specific α2 modulation of cytosolic Ca2+ signals suggests a novel retinal mechanism that could contribute significantly to the reduction of RGC injury associated with α2 agonist treatment in animal models of glaucoma and acute retinal ischemia. 
Retinal α2 Adrenergic System
Unlike many other neurotransmitter systems in the retina, such as glutamate, GABA, glycine, acetylcholine, and dopamine, whose physiological function in the retina has been studied extensively, the normal function of the retinal α2 adrenergic system is largely unknown, 17 18 even though compelling evidence indicates that α2 receptors are expressed in the retina. 13 14 15 On the other hand, strong evidence indicates that α2 agonists are neuroprotective in a wide range of models for injury to retinal neurons. For example, α2 agonists, such as brimonidine, can reduce RGC injury in various experimental retinal disease models, including acute retinal ischemia, 8 9 10 11 glaucoma, 14 and optic nerve injury. 12 However, the underlying mechanism(s) for this α2-mediated protection is (are) not well understood. 
It has been demonstrated in other neural tissues that presynaptic α2 receptors regulate transmitter release by modulating voltage-gated K+ and Ca2+ (N or P/Q subtypes) channels. 19 20 21 22 Our results not only provide the first direct evidence in the retina that the α2 mechanism modulates activity of voltage-gated Ca2+ channels, they suggest that this modulation is mainly on the L-type because the depolarization-elicited elevation of the cytosolic free Ca2+ is almost fully blocked by the selective L-type channel blocker nimodipine (Fig. 4) . The L-type Ca2+ channel is a major subtype in the retina that plays a critical role in neurotransmitter release from photoreceptors and from bipolar cells. 18 23 Given that α2 modulation occurred only at IPL, our results suggest that a normal function of the α2 mechanism in the retina is the modulation of synaptic transmission between second- and third-order retinal neurons. Under disease conditions, this negative modulation of Ca2+ influx (which would be expected to limit the release of neurotransmitters, particularly glutamate) may significantly reduce glutamatergic excitotoxic injury to RGCs. Consistent with this is a previous finding that, in acute retinal ischemia, brimonidine treatment not only reduces ischemic injury to RGCs, it significantly reduces ischemia-induced elevation of vitreal glutamate levels. 9  
Abnormal elevation of cytosolic free Ca2+, produced mainly by Ca2+ influx through voltage- or ligand-gated Ca2+ channels, or both, has been demonstrated to contribute significantly to neuronal injury in many neural disorders. 3 4 24 Therefore, it is conceivable that a direct negative modulation of depolarization-elicited Ca2+ influx in RGCs (in this case, the dendrites of RGCs) by α2 agonists may also contribute to neuroprotective activity. Indeed, previous studies have shown that α2 receptors are expressed in IPL and RGC layers. 13 14 15 Thus, the α2 system could protect RGCs under disease conditions by presynaptic (preventing the excess release of glutamate) and postsynaptic (preventing the overactivation of L-type Ca2+ channels on RGCs) mechanisms. More work is needed on specific types of retinal cells (such as bipolar cells and RGCs themselves) to identify directly these presynaptic and postsynaptic mechanisms. 
Primary and Secondary Ca2+ Signals
Our results suggest that the high K+-elicited increase of cytosolic Ca2+ predominantly represents Ca2+ influx through voltage-gated Ca2+ channels (particularly the L-type channels). Because membrane depolarization and voltage-gated Ca2+ channel activation could trigger the release of excitatory neurotransmitters such as glutamate, a secondary Ca2+ signal may be generated as a result of the activation of Ca2+-permeable ionotropic glutamate receptors (such as NMDA receptors, which are highly Ca2+ permeable and are sometimes called ligand-gated Ca2+ channels). However, direct testing with a selective NMDA receptor antagonist D-AP5 showed that this NMDA receptor–mediated secondary contribution was relatively small (Fig. 4C , right pair). In cultured rat retinal neurons, it has been shown that brimonidine attenuates a glutamate-elicited increase of cytosolic Ca++. 25 Therefore, a similar α2 modulation of NMDA receptor function could make a small contribution to the overall α2 modulation of Ca2+ signals at IPL. 
Region-Specific Modulation of Ca2+ Signals by α2 Agonists
It is surprising that α2 agonists selectively modulated Ca2+ signals at IPL but not at OPL, despite the fact that the Ca2+ signal at both synaptic layers was mediated mainly by L-type Ca2+ channels (Fig. 4) . α2 Receptors are expressed in outer retina, albeit at a significantly lower level. 15 One possible explanation is that α2 receptors in outer retina may not be coupled to the L-type Ca2+ channels but make use of a different intracellular signaling pathway. For example, it has been reported that α2 agonists are neuroprotective to photoreceptors. This effect is mediated by the induction of basic fibroblast growth factor, a unique α2-mediated action found in retinal photoreceptors but nowhere else in the CNS. 26 Alternatively, α2 agonists may have a differential (opposite) effect on L-type Ca2+ channels at rod and cone terminals, which could explain the absence of an α2 effect on the overall Ca2+ signal at OPL. This kind of differential effect of dopamine and somatostatin has been reported earlier by others. 27 28  
Although functional NMDA receptors are expressed predominantly, if not exclusively, on third-order retinal neurons, this differential expression pattern cannot be the main reason for this region-specific modulation of Ca2+ signals by α2 agonists because NMDA receptor contribution to the overall high K+-elicited Ca2+ signal at IPL is relatively small (Fig. 4C)
Conclusions
Our findings have provided the first evidence that α2 receptors can modulate the activity of L-type Ca2+ channels in the CNS. They also show that this α2 modulation is region specific in the retina in that it occurs at the IPL but not at the OPL. These findings suggest that a physiological function of the retinal α2 system is the regulation of synaptic transmission at IPL and that brimonidine and other α2 agonists may protect RGCs from experimental injury by preventing abnormal elevation of cytosolic free Ca2+ either in RGCs, in their presynaptic cells, or in both. 
 
Figure 1.
 
Functional Ca2+ imaging in living rat retinal slice. (A) Bright-field image of a representative living rat retinal slice. The yellow rectangle encompasses the area in which the Ca2+ signal (B) was measured. (B) High K+-elicited elevation of cytosolic free Ca2+ at IPL. This high K+-elicited Ca2+ signal was abolished during perfusion of 0 Ca2+ Ringer. The y-axis is normalized fluorescent intensity. The black horizontal bar indicates the duration of local perfusion with high K+ Ringer. (C) Statistical data for the effects of 0 Ca2+ (2 ± 3; n = 7) and 10 μM nimodipine (8 ± 21; n = 4) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 1.
 
Functional Ca2+ imaging in living rat retinal slice. (A) Bright-field image of a representative living rat retinal slice. The yellow rectangle encompasses the area in which the Ca2+ signal (B) was measured. (B) High K+-elicited elevation of cytosolic free Ca2+ at IPL. This high K+-elicited Ca2+ signal was abolished during perfusion of 0 Ca2+ Ringer. The y-axis is normalized fluorescent intensity. The black horizontal bar indicates the duration of local perfusion with high K+ Ringer. (C) Statistical data for the effects of 0 Ca2+ (2 ± 3; n = 7) and 10 μM nimodipine (8 ± 21; n = 4) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 2.
 
Suppression by brimonidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 600 nM brimonidine on the Ca2+ signal. (B) Dose-response relationship of brimonidine effect on the Ca2+ signal at IPL. (C) The brimonidine effect is blocked by the α2 receptor antagonist yohimbine. (D) Statistical data for the effects of 600 nM brimonidine (0.1 ± 0.8; n = 6) and two α2 receptor antagonists, 10 μM yohimbine (98 ± 4; n = 6) and 10 μM rauwolscine (91 ± 9; n = 6) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 2.
 
Suppression by brimonidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 600 nM brimonidine on the Ca2+ signal. (B) Dose-response relationship of brimonidine effect on the Ca2+ signal at IPL. (C) The brimonidine effect is blocked by the α2 receptor antagonist yohimbine. (D) Statistical data for the effects of 600 nM brimonidine (0.1 ± 0.8; n = 6) and two α2 receptor antagonists, 10 μM yohimbine (98 ± 4; n = 6) and 10 μM rauwolscine (91 ± 9; n = 6) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 3.
 
Suppression by medetomidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 300 nM medetomidine (control for yohimbine group: 32 ± 14, n = 7; control for atipamezole group: 37 ± 19, n = 6) applied alone or in combination with 10 μM yohimbine (79 ± 12; n = 7) or 3 μM atipamezole (98 ± 9; n = 6). (B) Dose-response relationship for medetomidine effect (blue) on the Ca2+ signal at IPL. Statistical numbers at each concentration are: 0.03 μM: 84 ± 6, n = 7; 0.1 μM: 63 ± 9, n = 9; 0.3 μM: 34 ± 16, n = 13; 0.6 μM: 28 ± 19, n = 7. For comparison, the dose-response curve for brimonidine (red, from Fig. 2B ) is also plotted. Data are plotted as mean ± SD. The two sets of dashed lines indicate the approximate IC50 for medetomidine (100 nM) and brimonidine (approximately 260 nM), respectively.
Figure 3.
 
Suppression by medetomidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 300 nM medetomidine (control for yohimbine group: 32 ± 14, n = 7; control for atipamezole group: 37 ± 19, n = 6) applied alone or in combination with 10 μM yohimbine (79 ± 12; n = 7) or 3 μM atipamezole (98 ± 9; n = 6). (B) Dose-response relationship for medetomidine effect (blue) on the Ca2+ signal at IPL. Statistical numbers at each concentration are: 0.03 μM: 84 ± 6, n = 7; 0.1 μM: 63 ± 9, n = 9; 0.3 μM: 34 ± 16, n = 13; 0.6 μM: 28 ± 19, n = 7. For comparison, the dose-response curve for brimonidine (red, from Fig. 2B ) is also plotted. Data are plotted as mean ± SD. The two sets of dashed lines indicate the approximate IC50 for medetomidine (100 nM) and brimonidine (approximately 260 nM), respectively.
Figure 4.
 
Region-specific modulation by α2 agonists of high K+-elicited Ca2+ signals. High K+ elicited a robust cytosolic Ca2+ increase at both OPL (A) and IPL (B) at the same retinal slices (n = 7). (C) Effects of 0 Ca2+, nimodipine (a selective L-type, voltage-gated Ca2+ channel blocker), brimonidine, and D-AP5 (a selective NMDA receptor antagonist) on high K+-elicited Ca2+ signals at OPL and IPL. Perfusion with 0 Ca2+ Ringer abolished the Ca2+ response at both OPL (2% ± 2%; n = 7) and IPL (2% ± 3%; n = 7). Application of 10 μM nimodipine also substantially attenuated the Ca2+ signal at both OPL (14% ± 18%; n = 4) and IPL (8% ± 21%; n = 4). Brimonidine 300 nM selectively suppressed the Ca2+ signal at IPL but not at OPL in the same retinal slices (IPL, 41% ± 12%; OPL, 95% ± 8%; n = 6). D-AP5 50 μM had a small but statistically significant effect (83% ± 6%, n = 4, P = 0.01, paired t-test) at IPL. It did not affect the Ca2+ signal at OPL (96% ± 4%, n = 4, P = 0.12). Data are plotted as mean ± SD.
Figure 4.
 
Region-specific modulation by α2 agonists of high K+-elicited Ca2+ signals. High K+ elicited a robust cytosolic Ca2+ increase at both OPL (A) and IPL (B) at the same retinal slices (n = 7). (C) Effects of 0 Ca2+, nimodipine (a selective L-type, voltage-gated Ca2+ channel blocker), brimonidine, and D-AP5 (a selective NMDA receptor antagonist) on high K+-elicited Ca2+ signals at OPL and IPL. Perfusion with 0 Ca2+ Ringer abolished the Ca2+ response at both OPL (2% ± 2%; n = 7) and IPL (2% ± 3%; n = 7). Application of 10 μM nimodipine also substantially attenuated the Ca2+ signal at both OPL (14% ± 18%; n = 4) and IPL (8% ± 21%; n = 4). Brimonidine 300 nM selectively suppressed the Ca2+ signal at IPL but not at OPL in the same retinal slices (IPL, 41% ± 12%; OPL, 95% ± 8%; n = 6). D-AP5 50 μM had a small but statistically significant effect (83% ± 6%, n = 4, P = 0.01, paired t-test) at IPL. It did not affect the Ca2+ signal at OPL (96% ± 4%, n = 4, P = 0.12). Data are plotted as mean ± SD.
QuigleyHA. Neuronal death in glaucoma. Prog Retinal Eye Res. 1999;18:39–57. [CrossRef]
DowlingJE. The Retina: An Approachable Part of the Brain. 1987;Harvard University Press Cambridge, MA.
TymianskiM, TatorCH. Normal and abnormal calcium homeostasis in neurons a basis for the pathophysiology of traumatic and ischemic central nervous system injury. Neurosurgery. 1996;38:1176–1195. [PubMed]
AgrawalSK, NashmiR, FehlingsMG. Role of L- and N-type calcium channels in the pathophysiology of traumatic spinal cord white matter injury. Neuroscience. 2000;99:179–188. [CrossRef] [PubMed]
SmithIF, GreenKN, LeFerlaFM. Calcium dysregulation in Alzheimer’s disease: recent advances gained from genetically modified animals. Cell Calcium. 2005;38:427–437. [CrossRef] [PubMed]
DochertyJR. Subtypes of functional α1- and α2-adrenoceptors. Eur J Pharmacol. 1998;361:1–15. [CrossRef] [PubMed]
WoldeMussieE, RuizG, WijonoM, WheelerL. Neuroprotection of retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular hypertension. Invest Ophthalmol Vis Sci. 2001;42:2849–2855. [PubMed]
ChaoHM, ChidlowG, MelenaJ, WoodJP, OsborneNN. An investigation into the potential mechanisms underlying the neuroprotective effect of clonidine in the retina. Brain Res. 2000;877:47–57. [CrossRef] [PubMed]
DonelloJE, PadilloEU, WebsterML, WheelerLA, GilDW. α2-Adrenoceptor agonists inhibit vitreal glutamate and aspartate accumulation and preserve retinal function after transient ischemia. J Pharmacol Exp Ther. 2001;296:216–223. [PubMed]
LafuenteMP, Villegas-PerezMP, Sobrado-CalvoP, Garcia-AvilesA, Miralles de ImperialJ, Vidal-SanzM. Neuroprotective effects of alpha-2 adrenergic agonists against ischemia-mediated retinal ganglion cell death. Invest Ophthalmol Vis Sci. 2001;42:2074–2084. [PubMed]
LaiRK, ChunT, HassonD, LeeS, MehrbodF, WheelerL. Alpha-2 adrenoceptor agonist protects retinal function after acute retinal ischemic injury in the rat. Vis Neurosci. 2002;19:175–185. [PubMed]
YolesE, WheelerLA, SchwartzM. Alpha2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration. Invest Ophthalmol Vis Sci. 1999;40:65–73. [PubMed]
ZarbinMA, WamsleyJK, PalaciosJM, KuharMJ. Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina. Brain Res. 1986;374:75–92. [CrossRef] [PubMed]
WheelerL, WoldeMussieE. Alpha-2 adrenergic receptor agonists are neuroprotective in experimental models of glaucoma. Eur J Ophthalmol. 2001;11:S30–S35. [PubMed]
KalapesiFB, CoroneoMT, HillMA. Human ganglion cells express the alpha-2 adrenergic receptor: relevance to neuroprotection. Br J Ophthalmol. 2005;89:758–763. [CrossRef] [PubMed]
WerblinFS. Transmission along and between rods in the tiger salamander retina. J Physiol (Lond). 1978;280:449–470. [CrossRef] [PubMed]
EhingerB, DowlingJE. Retinal neurocircuitry and transmission.BjorklundA HokfeltT SwansonLW eds. Handbook of Chemical Neuroanatomy Vol. 5: Integrated Systems of the CNS: Part 1. 1987;Elsevier Science Publishers BV Amsterdam, The Netherlands.
HeidelbergerR, ThoresonWB, WitkovskyP. Synaptic transmission at retinal ribbon synapses. Prog Retinal Eye Res. 2005;24:682–720. [CrossRef]
BoehamS, HuckS. Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release. Eur J Neurosci. 1996;8:1924–1931. [CrossRef] [PubMed]
DeBockF, KurzJ, AzadSC, et al. Alpha 2-adrenoceptor activation inhibits LTP and LTD in the basolateral amygdala: involvement of Gi/o-protein-mediated modulation of Ca2+ channels and inwardly rectifying K+ channels in LTD. Eur J Neurosci. 2003;17:1411–1424. [CrossRef] [PubMed]
MillerRJ. Presynaptic receptors. Annu Rev Pharmacol Toxicol. 1998;38:201–207. [CrossRef] [PubMed]
TimmonsSD, GeisertE, StewartAE, LorenzonNM, FoehringRC. Alpha2-adrenergic receptor-mediated modulation of calcium current in neocortical pyramidal neurons. Brain Res. 2004;1014:184–196. [CrossRef] [PubMed]
AkopianA, WitkovskyP. Calcium and retinal function. Mol Neurobiol. 2002;25:113–132. [CrossRef] [PubMed]
LuYM, YinHZ, ChiangJ, WeissJH. Ca2+-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury. J Neurosci. 1996;16:5457–5465. [PubMed]
BapitsteDC, HartwickAT, JollimoreCA, et al. Comparison of the neuroprotective effects of adrenoceptor drugs in retinal cell culture and intact retina. Invest Ophthalmol Vis Sci. 2002;43:2666–2676. [PubMed]
WenR, ChengT, LiY, CaoW, SteinbergRH. Alpha 2-adrenergic agonists induce basic fibroblast growth factor in photoreceptors in vivo and ameliorate light damage. J Neurosci. 1996;16:5986–5992. [PubMed]
StellaSL, Jr, ThoresonWB. Differential modulation of rod and cone calcium currents in tiger salamander retina by D2 dopamine receptors and cAMP. Eur J Neurosci. 2000;12:3537–3548. [CrossRef] [PubMed]
AkopianA, JohnsonJ, GabrielR, BrechaN, WitkovskyP. Somatostatin modulates voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors of the salamander retina. J Neurosci. 2000;20:929–936. [PubMed]
Figure 1.
 
Functional Ca2+ imaging in living rat retinal slice. (A) Bright-field image of a representative living rat retinal slice. The yellow rectangle encompasses the area in which the Ca2+ signal (B) was measured. (B) High K+-elicited elevation of cytosolic free Ca2+ at IPL. This high K+-elicited Ca2+ signal was abolished during perfusion of 0 Ca2+ Ringer. The y-axis is normalized fluorescent intensity. The black horizontal bar indicates the duration of local perfusion with high K+ Ringer. (C) Statistical data for the effects of 0 Ca2+ (2 ± 3; n = 7) and 10 μM nimodipine (8 ± 21; n = 4) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 1.
 
Functional Ca2+ imaging in living rat retinal slice. (A) Bright-field image of a representative living rat retinal slice. The yellow rectangle encompasses the area in which the Ca2+ signal (B) was measured. (B) High K+-elicited elevation of cytosolic free Ca2+ at IPL. This high K+-elicited Ca2+ signal was abolished during perfusion of 0 Ca2+ Ringer. The y-axis is normalized fluorescent intensity. The black horizontal bar indicates the duration of local perfusion with high K+ Ringer. (C) Statistical data for the effects of 0 Ca2+ (2 ± 3; n = 7) and 10 μM nimodipine (8 ± 21; n = 4) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 2.
 
Suppression by brimonidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 600 nM brimonidine on the Ca2+ signal. (B) Dose-response relationship of brimonidine effect on the Ca2+ signal at IPL. (C) The brimonidine effect is blocked by the α2 receptor antagonist yohimbine. (D) Statistical data for the effects of 600 nM brimonidine (0.1 ± 0.8; n = 6) and two α2 receptor antagonists, 10 μM yohimbine (98 ± 4; n = 6) and 10 μM rauwolscine (91 ± 9; n = 6) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 2.
 
Suppression by brimonidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 600 nM brimonidine on the Ca2+ signal. (B) Dose-response relationship of brimonidine effect on the Ca2+ signal at IPL. (C) The brimonidine effect is blocked by the α2 receptor antagonist yohimbine. (D) Statistical data for the effects of 600 nM brimonidine (0.1 ± 0.8; n = 6) and two α2 receptor antagonists, 10 μM yohimbine (98 ± 4; n = 6) and 10 μM rauwolscine (91 ± 9; n = 6) on IPL Ca2+ signal. Data are plotted as mean ± SD.
Figure 3.
 
Suppression by medetomidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 300 nM medetomidine (control for yohimbine group: 32 ± 14, n = 7; control for atipamezole group: 37 ± 19, n = 6) applied alone or in combination with 10 μM yohimbine (79 ± 12; n = 7) or 3 μM atipamezole (98 ± 9; n = 6). (B) Dose-response relationship for medetomidine effect (blue) on the Ca2+ signal at IPL. Statistical numbers at each concentration are: 0.03 μM: 84 ± 6, n = 7; 0.1 μM: 63 ± 9, n = 9; 0.3 μM: 34 ± 16, n = 13; 0.6 μM: 28 ± 19, n = 7. For comparison, the dose-response curve for brimonidine (red, from Fig. 2B ) is also plotted. Data are plotted as mean ± SD. The two sets of dashed lines indicate the approximate IC50 for medetomidine (100 nM) and brimonidine (approximately 260 nM), respectively.
Figure 3.
 
Suppression by medetomidine of high K+-elicited Ca2+ signal at IPL. (A) Effect of 300 nM medetomidine (control for yohimbine group: 32 ± 14, n = 7; control for atipamezole group: 37 ± 19, n = 6) applied alone or in combination with 10 μM yohimbine (79 ± 12; n = 7) or 3 μM atipamezole (98 ± 9; n = 6). (B) Dose-response relationship for medetomidine effect (blue) on the Ca2+ signal at IPL. Statistical numbers at each concentration are: 0.03 μM: 84 ± 6, n = 7; 0.1 μM: 63 ± 9, n = 9; 0.3 μM: 34 ± 16, n = 13; 0.6 μM: 28 ± 19, n = 7. For comparison, the dose-response curve for brimonidine (red, from Fig. 2B ) is also plotted. Data are plotted as mean ± SD. The two sets of dashed lines indicate the approximate IC50 for medetomidine (100 nM) and brimonidine (approximately 260 nM), respectively.
Figure 4.
 
Region-specific modulation by α2 agonists of high K+-elicited Ca2+ signals. High K+ elicited a robust cytosolic Ca2+ increase at both OPL (A) and IPL (B) at the same retinal slices (n = 7). (C) Effects of 0 Ca2+, nimodipine (a selective L-type, voltage-gated Ca2+ channel blocker), brimonidine, and D-AP5 (a selective NMDA receptor antagonist) on high K+-elicited Ca2+ signals at OPL and IPL. Perfusion with 0 Ca2+ Ringer abolished the Ca2+ response at both OPL (2% ± 2%; n = 7) and IPL (2% ± 3%; n = 7). Application of 10 μM nimodipine also substantially attenuated the Ca2+ signal at both OPL (14% ± 18%; n = 4) and IPL (8% ± 21%; n = 4). Brimonidine 300 nM selectively suppressed the Ca2+ signal at IPL but not at OPL in the same retinal slices (IPL, 41% ± 12%; OPL, 95% ± 8%; n = 6). D-AP5 50 μM had a small but statistically significant effect (83% ± 6%, n = 4, P = 0.01, paired t-test) at IPL. It did not affect the Ca2+ signal at OPL (96% ± 4%, n = 4, P = 0.12). Data are plotted as mean ± SD.
Figure 4.
 
Region-specific modulation by α2 agonists of high K+-elicited Ca2+ signals. High K+ elicited a robust cytosolic Ca2+ increase at both OPL (A) and IPL (B) at the same retinal slices (n = 7). (C) Effects of 0 Ca2+, nimodipine (a selective L-type, voltage-gated Ca2+ channel blocker), brimonidine, and D-AP5 (a selective NMDA receptor antagonist) on high K+-elicited Ca2+ signals at OPL and IPL. Perfusion with 0 Ca2+ Ringer abolished the Ca2+ response at both OPL (2% ± 2%; n = 7) and IPL (2% ± 3%; n = 7). Application of 10 μM nimodipine also substantially attenuated the Ca2+ signal at both OPL (14% ± 18%; n = 4) and IPL (8% ± 21%; n = 4). Brimonidine 300 nM selectively suppressed the Ca2+ signal at IPL but not at OPL in the same retinal slices (IPL, 41% ± 12%; OPL, 95% ± 8%; n = 6). D-AP5 50 μM had a small but statistically significant effect (83% ± 6%, n = 4, P = 0.01, paired t-test) at IPL. It did not affect the Ca2+ signal at OPL (96% ± 4%, n = 4, P = 0.12). Data are plotted as mean ± SD.
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